I. Field of the Invention
The present invention concerns amino acid sequence variants of hepatocyte growth factor (HGF), methods and means for preparing such variants, and pharmaceutical compositions comprising them.
II. Description of Background and Related Art
HGF was identified initially as a mitogen for hepatocytes [Michalopoulos et al., Cancer Res. 44, 4414-4419 (1984); Russel et al., J. Cell. Physiol. 119, 183-192 (1984) and Nakamura et al., Biochem. Biophys. Res. Comm. 122:1450-1459 (1984)]. Nakamura et al., Supra reported the purification of HGF from the serum of partially hepatectomized rats. Subsequently, HGF was purified from rat platelets, and its subunit structure was determined [Nakamura et al., Proc. Natl. Acad. Sci. U.S.A., 83, 6489-6493 (1986); and Nakamura et al., FEBS Letters 224, 311-316 (1987)]. The purification of human HGF (huHGF) from human plasma was first described by Gohda et al., J. Clin. Invest. 81, 414-419 (1988).
Both rat HGF and huHGF have been molecularly cloned, including the cloning and sequencing of a naturally occurring variant lacking 5 amino acids designated "delta5 HGF" [Miyazawa et al., Biochem. Biophys. Res. Comm. 163, 967-973 (1989); Nakamura et al., Nature 342, 440-443 (1989); Seki et al, Biochem. and Biophys. Res. Commun. 172, 321-327 (1990); Tashiro et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3200-3204 (1990); Okajima et al., Eur. J. Biochem. 193, 375-381 (1990)].
The mature form of huHGF, corresponding to the major form purified from human serum, is a disulfide linked heterodimer derived by proteolytic cleavage of the human pro-hormone (SEQ ID 22) between amino acids R494 and V495. This cleavage process generates a molecule composed of an .alpha.-subunit of 440 amino acids (M.sub.r 69 kDa) and a .beta.-subunit of 234 amino acids (M.sub.r 34 kDa). The nucleotide sequence of the hHGF cDNA reveals that both the .alpha.- and the .beta.-chains are contained in a single open reading frame coding for a pre-pro precursor protein. In the predicted primary structure of mature hHGF, an interchain S-S bridge is formed between Cys 487 of the .alpha.-chain and Cys 604 in the .beta.-chain (see Nakamura et al., Nature, supra). The N-terminus of the .alpha.-chain is preceded by 54 amino acids, starting with a methionine group. This segment includes a characteristic hydrophobic leader (signal) sequence of 31 residues and the prosequence. The .alpha.-chain starts at amino acid (aa) 55, and contains four Kringle domains. The so called "hairpin domain" includes amino acid residues 70-96 of wild-type human HGF. The Kringle 1 domain extends from about aa 128 to about aa 206, the Kringle 2 domain is between about aa 211 and about aa 288, the Kringle 3 domain is defined as extending from about aa 303 to about aa 383, and the Kringle 4 domain extends from about aa 391 to about aa 464 of the .alpha.-chain. It will be understood that the definition of the various Kringle domains is based on their homology with kringle-like domains of other proteins (prothrombin, plasminogen), therefore, the above limits are only approximate. As yet, the function of these Kringles has not been determined. The .beta.-chain of huHGF shows high homology to the catalytic domain of serine proteases (38% homology to the plasminogen serine protease domain). However, two of the three residues which form the catalytic triad of serine proteases are not conserved in huHGF. Therefore, despite its serine protease-like domain, hHGF appears to have no proteolytic activity and the precise role of the .beta.-chain remains unknown. HGF contains four putative glycosylation sites, which are located at positions 294 and 402 of the .alpha.-chain and at positions 566 and 653 of the .beta.-chain.
In a portion of cDNA isolated from human leukocytes in-frame deletion of 15 base pairs was observed. Transient expression of the cDNA sequence in COS-1 cells revealed that the encoded HGF molecule (delta5 HGF) lacking 5 amino acids in the Kringle 1 domain was fully functional (Seki et al., supra).
A naturally occurring huHGF variant has recently been identified which corresponds to an alternative spliced form of the huHGF transcript containing the coding sequences for the N-terminal finger and first two kringle domains of mature huHGF [Chan et al., Science 254, 1382-1385 (1991); Miyazawa et al., Eur. J. Biochem, 197, 15-22 (1991)]. This variant, designated HGF/NK2, has been proposed to be a competitive antagonist of mature huHGF.
The comparison of the amino acid sequence of rat HGF with that of huHGF revealed that the two sequences are highly conserved and have the same characteristic structural features. The length of the four Kringle domains in rat HGF is exactly the same as in huHGF. Furthermore, the cysteine residues are located in exactly the same positions; an indication of similar three-dimensional structures (Okajima et al., supra; Tashiro et al., supra).
The HGF receptor has been identified as the product of the c-Met proto-oncogene [Bottaro et al., Science 251, 802-804 (1991); Naldini et al., Oncogene 6, 501-504 (1991)], an 190-kDa hererodimeric (a disulfide-linked 50-kDa .alpha.-chain and a 145-kDa .beta.-chain) membrane-spanning tyrosine kinase protein [Park et al., Proc. Natl. Acad. Sci. U.S.A. 84, 6379-6383 (1987)]. The c-Met protein becomes phosphorylated on tyrosine residues of the 145-kDa .beta.-subunit upon HGF binding.
The levels of HGF increase in the plasma of patients with hepatic failure (Gohda et al., supra) and in the plasma [Lindroos et al., Hepatol. 13, 734-750 (1991)] or serum [Asami et al,, J. Biochem. 109, 8-13 (1991)] of animals with experimentally induced liver damage. The kinetics of this response is rapid, and precedes the first round of DNA synthesis during liver regeneration suggesting that HGF may play a key role in initiating this process. More recently, HGF has been shown to be a mitogen for a variety of cell types including melanocytes, renal tubular cells, keratinocytes, certain endothelial cells and cells of epithelial origin [Matsumoto et al., Biochem. Biophys. Res. Commun. 176, 45-51 (1991); Igawa et al., Biochem. Biophys. Res. Commun. 174, 831-838 (1991); Han et al., Biochem. 30, 9768-9780 (1991); Rubin et al., Proc. Natl. Acad. Sci. U.S.A. 88, 415-419 (1991)]. Interestingly, HGF can also act as a "scatter factor", an activity that promotes the dissociation of epithelial and vascular endothelial cells in vitro [Stoker et al., Nature 327, 239-242 (1987); Weidner et al., J. Cell Biol. 111, 2097-2108 (1990); Naldini et al., EMBO J. 10, 2867-2878 (1991)]. Moreover, HGF has recently been described as an epithelial morphogen [Montesano et al., Cell 67, 901-908 (1991)]. Therefore, HGF has been postulated to be important in tumor invasion and in embryonic development. Chronic c-Met/HGF receptor activation has been observed in certain malignancies [Cooper et al., EMBO J. 5, 2623 (1986); Giordano et al., Nature 339, 155 (1989)].
It would be desirable to better understand the structure-activity relationship of HGF in order to identify functionally important domains in the HGF amino acid sequence.
It would be particularly desirable to identify the amino acid residues which are responsible for the interaction of HGF with its receptor.
It would be also desirable to identify the amino acid residues which are responsible for HGF biological activity.
It would further be desirable to provide amino acid sequence variants of HGF that have altered (preferably enhanced) receptor binding affinity as compared to the corresponding mature, wild-type HGF.
It would also be desirable to provide HGF amino acid sequence variants which have retained or enhanced receptor binding affinity as compared to the corresponding wild-type HGF, but are substantially devoid of HGF biological activity. Such molecules could act as competitive antagonists of HGF action.
It would further be desirable to provide HGF amino acid sequence variants that have retained or enhanced receptor binding affinity and increased biological activity as compared to the corresponding wild-type HGF (HGF agonists). Accordingly, it is an object of the present invention to provide HGF variants having retained or improved the receptor binding affinity of the corresponding mature wild-type HGF. It is another object of the invention to provide HGF variants that have retained substantially full receptor binding affinity of the corresponding mature wild-type HGF and are substantially incapable of HGF receptor activation. It is a further object to provide HGF variants that have retained substantially full receptor binding affinity of the corresponding mature wild-type HGF and Have improved biological properties.
These and further objects will be apparent to one of ordinary skill in the art.